肠道病毒71型感染诱导细胞程序性死亡形态学及分子生物学特征的初步研究

Primary study on molecular characteristics and mechanisms of enterovirus type 71-induced programmed cell death

肠道病毒71型(enterovirus type 71,EV71)感染常可引起婴幼儿手足口病(hand,foot and mouth disease,HFMD),还可引起中枢神经系统并发症等重症,甚至死亡。研究认为,EV71诱发重症的原因主要与病毒感染诱导细胞程序性死亡(programmed cell death,PCD)及诱导细胞产生大量炎症因子有关。病毒感染可通过激活不同的信号通路触发细胞程序性死亡,主要包括含半胱氨酸的天冬氨酸蛋白水解酶(cysteinyl aspartate specific proteinase,caspase)依赖的细胞凋亡、细胞焦亡,以及非caspase依赖的细胞坏死性凋亡。本研究旨在探讨EV71感染诱导细胞程序性死亡的形态学和分子生物学特征,利用显微镜和免疫荧光技术检测EV71感染后细胞形态变化,JC-1染色检测感染后细胞线粒体膜电位变化,流式细胞术及Annexin VFITC/PI双染法、乳酸脱氢酶释放量法检测感染细胞的细胞膜损伤程度,结合蛋白免疫印迹法检测病毒感染后细胞中多聚ADP核糖聚合酶[poly(ADP-ribose)polymerase,PARP]、caspase-9、caspase-3等凋亡因子,以及细胞焦亡关键效应蛋白Gasdermin D、坏死性凋亡效应蛋白MLKL的磷酸化情况。结果显示,EV71感染后细胞主要呈现凋亡特征,并伴随少量细胞坏死。与细胞凋亡相关的PARP被剪切,caspase-9和caspase-3等相关因子被激活。经泛caspase抑制剂处理后,细胞程序性死亡被抑制,但仍有部分细胞坏死。结果提示,EV71感染以细胞凋亡为主,也可能存在非caspase依赖的细胞程序性死亡。

Enterovirus type 71 （EV71） infection usually causes hand, foot and mouth disease （HFMD） in infants and young children. Severe EV71 infections can result in central nervous system complications, and even death. Previous studies suggest that EV71-induced severe complications are associated with programmed cell death and production of pro-inflammatory factors. To determine the molecular morphology and characteristics of EV71-induced cell death, EV71-infected cells were subjected to morphological analysis, JC-1 staining for potential of mitochondrial membrane, fluorescence-activated cell sorting, Annexin V-FITC/PI double staining, and lactate dehydrogenase （LDH） release assay. Western blotting was used to identify the activation of programmed cell death factors, including poly （ADP-ribose） polymerase （PARP）, caspase-9, caspase-3, Gasdermin D, and MLKL. The results showed that EV71-infected cells mainly induced apoptotic cell death with a small portion of cellular necrosis. The cleavage of PARP, caspase-9, caspase-3 was observed. Although cell death was mostly inhibited by a caspase inhibitor, cell death still could be observed during the early stage of virus infection. The results suggest that EV71 infection mainly induces apoptotic cell death in combination with a caspase-independent cell programmed death.